Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds

Definitions

• the present invention relates to a process for the production of cellulose by microbiological means.
• the higher plants constitute the raw material from which industrial cellulose is obtained; it is one of the major constituents, since it generally represents 30 to 50% of the weight of these plants.
• the renewal period for these raw materials is several decades for trees and one year for cereals.
• cellulose In higher plants, cellulose is often, from a weight point of view, the most important constituent, but it is accompanied, in variable quantities, by lignins, hemicelluloses, pectic compounds and many other substances which are associated, or combined ("encrusted") with cellulose.
• the cost price of the cellulose obtained according to conventional processes is due, in a proportion of 60% to 80% to the cost: of energy, chemicals and industrial operations, necessary for the separation and the elimination of non-cellulosic substances, as well as the purification (including bleaching) of the cellulose itself, in particular with regard to the purified and semi-purified qualities of cellulose.
• the present invention due to the work of Mr. P. LABOUREUR relates to an industrial process for the manufacture of bacterial cellulose for all uses, mainly chemical, but also paper makers.
• the present invention relates to a process for producing cellulose of bacterial origin from materials of vegetable origin containing carbohydrates, in a submerged culture bioreactor -, - characterized in that in the production medium comprising said carbohydrates, a cellulose-producing bacterial strain is cultivated in the hands, in that said bioreactor comprises means allowing the fixing of microorganisms and the development of cellulose fibers and in that during the production phase cellulose, aeration is poor and 1 • stirring is maintained with - minimum shear to promote the development of cellulose fibers.
• the bacterial strain used as biosynthesis and / or bioconversion agent is preferably:
• a bacterium whose cellulose production is essentially exocellular.
• a bacterium which can develop under selective conditions.
• microorganisms studied for the biosynthesis of cellulose there may be mentioned unicellular algae with rapid growth, for example, of the genus Oocystis or Glaucocystis, lower fungi, in particular oomycetes, for example of the genera: Pythium, Phytophtora, Saprolegnia, Achyla , but for various reasons, these microorganisms seem difficult to use on an industrial scale (i).
• the production of cellulose, under industrial conditions, is the property of certain selected strains, but not of all strains of the same genus, the same species or subspecies.
• the method according to the present invention aims to allow production on an industrial scale.
• the process according to the invention for the industrial production of bacterial cellulose is carried out in submerged culture, in a bioreactor.
• Production can be obtained: - either in a single step (or operation), that is to say with simultaneously: development of the microorganism, biosynthesis and / or biconversion agent, and production of cellulose, - - ⁇ • either in two stages (of the operations) with development of the microorganism agent of biosynthesis and / or biconversion, in a first stage, then separation of this microorganism, and in a second stage production of cellulose with non-proliferating microorganism.
• This second technique is similar to bioconversions.
• a process that would use not the entire microbial reagent, but a fraction subcellular containing the enzymatic systems and cofactors necessary for the transformation of the carbonaceous substrate into cellulose can also be envisaged.
• the method according to the invention is based on highlighting the characteristics necessary for a submerged culture. As indicated, the aeration must remain weak during the production phase, for example less than 1.5 ppm, preferably less than 0.5 ppm -t'O j dissolved in the reactor.
• the agitation must be low, this parameter is difficult to define because it depends on the characteristics of the reactor, and those of the agitation system and also on the parameters of the medium.
• Optimal agitation can nevertheless be determined by a person skilled in the art. It is important that the stirring system is adjustable during cultivation, and can be such that, during the cellulose production phase, there is no turbulence and / or shearing effect which would prevent the formation of cellulose, then its polymerization, and its crystallization and the organization of fibrils; excessive agitation has negative effects; at the end of fermentation a simple slow brewing is preferable.
• cellulose fibers may be supports of various shapes or other devices suitable for the development of cellulose fibers, step by step, from the fixing sites. In fact, we have been able to demonstrate that cellulose does not form very difficult in the free liquid phase, without fixing sites.
• the composition of the production medium may be as follows: carbon substrate 20 g / 1 to 150 g / 1 (see below) preferably. 30 g / 1 to 60 g / 1 citric acid 0-, 2- g / 1 to 6 g / 1, preferably 0.5 g / 1 to 3 g / 1 yeast extract 0.1 g / 1- to 2 g / 1 preferably 0-, 5 g / l__ at * 1 g / 1 or corn steep "** NH4C1 2 g / 1 to 10 g / 1 preferably-3 g / 1 to 6 g / 1 or (NH4) 2 S04or N NHH 44 NN00 33 or urea HK2 P04 0.2 g / 1 to 0.66 g / 1 preferred 0.3 g / 1 to 0.5 g / 1 (see below) preferably. 30 g / 1 to 60 g / 1 citric acid 0-, 2- g / 1 to 6 g / 1, preferably
• H aC03 0.2 g / 1 to 0.8 g / 1 preferred.
• 0.3 g / 1 to 0.6 g / 1 CaC12 0.5 g / 1 to 2 g / 1 preferably 0.7 g / 1 to 5 g / 1 pH 4.5 to 5.5 preferably from 4.7 to 4.9 sterilization 10 'to 20' to 110 'to 120' preferably 15 'to 115' -
• the medium will preferably be maintained at a pH below pH5, sterilization can thus be eliminated. Notes on the production environment
• the nature of the carbonaceous substrate depends on the biochemical characteristics of the microbial strain - used in particular of its enzymatic equipment, which allows it or not, to use the carbonaceous substrate.
• the recommended carbon substrates are: carbohydrates of vegetable origin which are made up or which contain hexoses more particularly glucose and fructose or possibly other hexoses or pentoses:
• sucrose with invertase + strains
• sucrose in its various forms: beet or cane juice, molasses, syrups, brown sugar, white sugar, glucose-fructose mixtures, for example; in certain cases, it may be useful to hydrolyze the raw materials containing sucrose for the invertase (-) strains and / or to make other carbohydrates usable;
• starches with amylase + strains
• derivatives flours, amylodextrins-dextrins, maltose, glucose, for example.
• hanolthanol or glycerol, or lactate or acetate or manitol which can stimulate the production of cellulose, as well as possibly methanol.
• the concentration of carbonaceous substrate, carbohydrate, in the production medium, such as sucrose or starch, is of great importance. This concentration must be between 20 and
• the production of cellulose is optimal between 35 and 65 g / 1 it is low from 25 g / 1, zero at lower concentration.
• the concentration of carbohydrates should preferably be maintained between 30 g / l and 50 g / l for Acetobacter at least during the cellulose production phase.
• citric acid has a beneficial role with certain microorganisms, for example certain strains of Acetobacter.
• magnesium it is very important for the production of cellulose for example with certain strains of Autobacter. e calcium and iron are favorable for certain strains.
• the other mineral elements and trace elements are generally provided in sufficient quantity by the impurities.
• Vitamin and growth factor requirements in particular Pab, Bl, B2, B6, B12, ac. folic and possibly in ac. amines (ac. glutamic, lysine, etc.) are covered by: - yeast extracts,
• the fermentation process must take account of the above characteristics.
• the production bioreactor is sown with a preculture aged 3 to 12 days, preferably 5 to 8 days.
• This preculture may, depending on the volume of the production bioreactor, be preceded by one or more other precultures of increasing volumes.
• the preculture (s) are made from a static culture on maintenance medium (in tubes).
• the maintenance cut, the preculture (s) are carried out, in a liquid medium, of the same composition, at the same pH and at the same temperature, as the production medium.
• Maintenance cultures should preferably be transplanted every 20 to 30 days.
• Stock cultures can be freeze-dried.
• the production bioreactor is sown with 5 to 20% by volume of preculture, preferably with 10%.
• the culture conditions, as well as the culture media depend on the strains used. With Acetobacter, the temperature is maintained between 25 * and 35 ', preferably between 28 * and 32 *, if possible between 29 * and 31 *, with other bacteria, the temperature may be different.
• the pH of the culture is maintained between 4.5 and 5.5, preferably between 4.6 and 4.8. With other bacteria, the pH of the culture may be different.
• the agitation expressed in revolutions / minute, variable according to the agitation system, must be controlled and limited, for example between 60 and 90 revolutions / minute, during the development phase of the bacteria and for example between 30 and 60 revolutions / minute during the cellulose production phase.
• the optimal conditions for agitation depend on the strain used and on the characteristics of the bioreactor and those of the agitation-aeration device.
• the aeration (expressed in volume of air, by volume of medium, per minute: Wm) variable according to the aeration system, must be controlled and limited, for example equal to or less than 1 Wm during the development phase of bacteria and for example equal to or lower than 0.5 Wm for the cellulose production.
• the nitrogen and phosphorus contents can be used to control and limit the growth of the biomass, for example during the cellulose production stage.
• the medium can be supplemented with carbon substrate 1 or more times, in particular when the critical minimum concentration is reached below which there is no longer any synthesis of cellulose. It may be advantageous, to accelerate the development, to use a low initial concentration of carbonaceous substrate, for example 10 to 20 g / l, to subsequently increase it, during the cellulose production phase.
• the medium can be recycled after separation of the cellulose and the biomass.
• the average duration of culture is generally between 40 and 120 h, preferably between 45 and 60 hours. However, this duration can be reduced by using a larger inoculum.
• D.P. degree of polymerization
• - increases with the age of the culture
• the method according to the invention allows simple purification of the cellulose obtained.
• the cellulose and biomass mixture is separated by racking, and or filtration of the liquid phase; - the mixture is drained,
• the mixture is washed thoroughly, repeatedly with water, to physically entrain the maximum of biomass (bacteria) and impurities (soluble and insoluble products from the culture medium).
• the cellulose, washed with water, is purified by any physical, physicochemical or chemical treatment making it possible to desorb and / or lyse (or dissolve), then to eliminate and entrain, the microbial bodies and possibly others impurities, which are still on or between the cellulose fibers, after washing with water.
• the purification of the cellulose obtained is preferably carried out by treatment with an organic or mineral acid, preferably hot acetic acid. It is possible to combine this treatment with the use of surfactants.
• the cellulose after spinning and washing with water, is treated cold or preferably hot, at 100 ', for 0.5 to 2 hours, preferably for one hour with acetic acid, at the right rate. from 1 to 15% preferably - 5 to 8% by volume relative to the volume of hydrated cellulose (after spinning). After this treatment with acetic acid, the cellulose is washed thoroughly with water.
• the cellulose is neutralized with a dilute sodium hydroxide solution, then rinsed again with water.
• a treatment with sodium hydroxide 1 to 5 N After this treatment with sodium hydroxide, wash abundantly with water, wring and dry if necessary.
• acetic acid for the elimination of microbial bodies and impurities, can optionally be replaced by treatment with other organic or mineral acids, combined or not with suitable surfactants.
• Purified bacterial cellulose is obtained with this process and according to the strains and growing conditions. with yields which can be between 30% and 70%. Yield: weight of synthesized cellulose weight of carbonaceous substrate used and with a productivity of between 10 and 100 g / l / J. . -.__- • -— *.
• Purified bacterial cellulose; - is 1 'o-cellulose having a D.P. between 500 and 3500 generally between 1000 and 1500; the bacterial cellulose thus obtained does not contain any other polysaccharides, hemicelluloses, pectic compounds or lignins.
• the purified bacterial cellulose thus obtained, according to the invention can be used as chemical cellulose for the manufacture of cellulose derivatives, such as cellulose acetate, nitrocellulose, carboxymethylcellulose, hydroxyethylcellulose, methylcellulose, hydroxymethylcellulose, etc. and for papermaking uses, in particular as a mixture in pulp or for other uses.
• cellulose derivatives such as cellulose acetate, nitrocellulose, carboxymethylcellulose, hydroxyethylcellulose, methylcellulose, hydroxymethylcellulose, etc.
• papermaking uses in particular as a mixture in pulp or for other uses.
• Example 1 strain - Acetobacter aceti subsp.xylinum
• cellulose purified by treatment with acetic acid for 1 hour at 100 ° C .; 5% by volume, neutralized with soda, and washed, cellulose D.P.-1200

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• Organic Chemistry (AREA)
• Chemical & Material Sciences (AREA)
• Engineering & Computer Science (AREA)
• Life Sciences & Earth Sciences (AREA)
• Zoology (AREA)
• Wood Science & Technology (AREA)
• Health & Medical Sciences (AREA)
• General Engineering & Computer Science (AREA)
• General Chemical & Material Sciences (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Biotechnology (AREA)
• Biochemistry (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Microbiology (AREA)
• General Health & Medical Sciences (AREA)
• Genetics & Genomics (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Polysaccharides And Polysaccharide Derivatives (AREA)
• Agricultural Chemicals And Associated Chemicals (AREA)
• Food Preservation Except Freezing, Refrigeration, And Drying (AREA)

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Citations (2)

• 1987
  • 1987-05-26 FR FR8707387A patent/FR2615864B1/fr not_active Expired - Lifetime
• 1988
  • 1988-05-26 AT AT88904978T patent/ATE107962T1/de not_active IP Right Cessation
  • 1988-05-26 EP EP88904978A patent/EP0318543B1/de not_active Expired - Lifetime
  • 1988-05-26 WO PCT/FR1988/000266 patent/WO1988009381A1/fr active IP Right Grant
  • 1988-05-26 DE DE3850477T patent/DE3850477T2/de not_active Expired - Fee Related
• 1989
  • 1989-01-25 FI FI890368A patent/FI95145C/fi not_active IP Right Cessation
  • 1989-01-26 NO NO890337A patent/NO176282C/no unknown

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